Components Nominal concentration (μg mL-1) Precision Intra-day (n =5) Inter-day (n =5) Mean±SD (μg mL-1) Accuracy (%) RSD (%) Mean±SD (μg mL-1) Accuracy (%) RSD (%) gamabufotalin 6.25 6.23 ±0.03 99.67 0.47 6.24 ±0.01 99.86 0.21 25 25.08±0.01 100.3 0.04 24.95 ±0.09 99.78 0.37 100 100.04±0.06 100.04 0.06 100.00 ±0.45 100 0.45 arenobufagin 3.13 3.13±0.00 100.09 0.15 3.15 ±0.03 100.69 0.8 12.5 12.55±0.02 100.42 0.16 12.50 ±0.09 99.99 0.75 50 50.15±0.09 100.3 0.18 50.10 ±0.33 100.2 0.67 telocinobufagin 4.69 4.68±0.01 99.86 0.27 4.69 ±0.03 99.97 0.65 18.75 18.75±0.04 100.02 0.23 18.76 ±0.10 100.05 0.52 75 75.13±0.08 100.18 0.11 74.98 ±0.33 99.97 0.44 desacetylcinobufotalin 4.69 4.68±0.01 99.82 0.19 4.69 ±0.04 100.11 0.77 18.75 18.75±0.06 100 0.33 18.72 ±0.06 99.85 0.35 75 75.05±0.07 100.07 0.09 74.98 ±0.35 99.98 0.46 bufotalin 7.5 7.49±0.01 99.86 0.09 7.52 ±0.04 100.31 0.58 30 30.05±0.04 100.16 0.14 29.99 ±0.20 99.98 0.68 120 120.01±0.04 100.01 0.03 119.80 ±0.55 99.84 0.46 cinobufotalin 5.63 5.61±0.01 99.67 0.13 5.62 ±0.01 99.76 0.16 22.5 22.50±0.01 100.01 0.06 22.52 ±0.01 100.1 0.06 90 90.05±0.06 100.06 0.06 90.07 ±0.28 100.08 0.31 bufalin 9.38 9.37±0.01 99.94 0.13 9.36 ±0.04 99.77 0.47 37.5 37.54±0.05 100.11 0.13 37.55 ±0.08 100.12 0.2 150 150.10±0.07 100.06 0.05 150.23 ±0.42 100.15 0.28 cinobufagin 12.5 12.49±0.03 99.94 0.26 12.56 ±0.08 100.45 0.63 50 50.01±0.04 100.02 0.08 50.14 ±0.15 100.28 0.29 200 200.00±0.02 100 0.01 200.22 ±0.32 100.11 0.16 resibufogenin 14.06 14.06±0.04 99.97 0.3 14.08 ±0.04 100.18 0.28 56.25 56.24±0.06 99.98 0.11 56.29 ±0.21 100.08 0.37 225 224.86±0.08 99.94 0.04 225.23 ±0.82 100.1 0.37

Table 2: Precision and accuracy of the developed method.